RESUMEN
Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Histonas/genética , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Quinasa Tipo Polo 1RESUMEN
In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein-dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl-binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.
Asunto(s)
Cinetocoros , Huso Acromático , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismoRESUMEN
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.
Asunto(s)
Proteínas Cromosómicas no Histona/ultraestructura , Cinetocoros/fisiología , Cinetocoros/ultraestructura , Centrómero/fisiología , Proteína A Centromérica/metabolismo , Proteína A Centromérica/ultraestructura , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Citoesqueleto , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas Nucleares/metabolismoRESUMEN
Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.
Asunto(s)
Proteína Adaptadora de Señalización CRADD/metabolismo , Caspasa 2/metabolismo , Centrosoma/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Proteína Adaptadora de Señalización CRADD/genética , Caspasa 2/genética , Diferenciación Celular , Cisteína Endopeptidasas/genética , Daño del ADN , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In mitosis, for each daughter cell to inherit an accurate copy of the genome from the mother cell, sister chromatids in the mother cell must attach to microtubules emanating from opposite poles of the mitotic spindle, a process known as bi-orientation. A surveillance mechanism, termed the spindle assembly checkpoint (SAC), monitors the microtubule attachment process and can temporarily halt the separation of sister chromatids and the completion of mitosis until bi-orientation is complete. SAC failure results in abnormal chromosome numbers, termed aneuploidy, in the daughter cells, a hallmark of many tumours. The HORMA-domain-containing protein mitotic arrest deficient 2 (MAD2) is a subunit of the SAC effector mitotic checkpoint complex (MCC). Structural conversion from the open to the closed conformation of MAD2 is required for MAD2 to be incorporated into the MCC. In vitro, MAD2 conversion and MCC assembly take several hours, but in cells the SAC response is established in a few minutes. Here, to address this discrepancy, we reconstituted a near-complete SAC signalling system with purified components and monitored assembly of the MCC in real time. A marked acceleration in MAD2 conversion and MCC assembly was observed when monopolar spindle 1 (MPS1) kinase phosphorylated the MAD1-MAD2 complex, triggering it to act as the template for MAD2 conversion and therefore contributing to the establishment of a physical platform for MCC assembly. Thus, catalytic activation of the SAC depends on regulated protein-protein interactions that accelerate the spontaneous but rate-limiting conversion of MAD2 required for MCC assembly.
Asunto(s)
Biocatálisis , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas Mad2/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Cinética , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Proteínas Tirosina Quinasas/metabolismo , Huso Acromático/metabolismo , Factores de TiempoRESUMEN
The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising â¼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Cinetocoros/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Cromosómicas no Histona/química , Humanos , Proteínas de Microfilamentos/química , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Especificidad por SustratoRESUMEN
The inositol polyphosphate 5'-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.
Asunto(s)
Cilios/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Dineínas/metabolismo , Ratones , Microscopía Fluorescente , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Accurate chromosome segregation during mitosis requires precise coordination of various processes, such as chromosome alignment, maturation of proper kinetochore-microtubule (kMT) attachments, correction of erroneous attachments, and silencing of the spindle assembly checkpoint (SAC). How these fundamental aspects of mitosis are coordinately and temporally regulated is poorly understood. In this study, we show that the temporal regulation of kMT attachments by CLASP1, astrin and Kif2b is central to mitotic progression and chromosome segregation fidelity. In early mitosis, a Kif2b-CLASP1 complex is recruited to kinetochores to promote chromosome movement, kMT turnover, correction of attachment errors, and maintenance of SAC signalling. However, during metaphase, this complex is replaced by an astrin-CLASP1 complex, which promotes kMT stability, chromosome alignment, and silencing of the SAC. We show that these two complexes are differentially recruited to kinetochores and are mutually exclusive. We also show that other kinetochore proteins, such as Kif18a, affect kMT attachments and chromosome movement through these proteins. Thus, CLASP1-astrin-Kif2b complex act as a central switch at kinetochores that defines mitotic progression and promotes fidelity by temporally regulating kMT attachments.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteína B del Centrómero/genética , Proteína B del Centrómero/metabolismo , Cromosomas/metabolismo , Humanos , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.
Asunto(s)
Neoplasias , Nucleótidos , Humanos , Cinética , Proteínas ras/genética , Transducción de Señal , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.
Asunto(s)
Proteínas de Ciclo Celular , Dineínas Citoplasmáticas , Complejo Dinactina , Proteínas de Ciclo Celular/metabolismo , Dineínas Citoplasmáticas/metabolismo , Complejo Dinactina/metabolismo , Cinetocoros/metabolismo , Conformación ProteicaRESUMEN
Electron paramagnetic resonance (EPR) spectroscopy is an established technique to site-specifically monitor conformational changes of spin-labeled biomolecules. Emerging in-cell EPR approaches aiming to address spin-labeled proteins in their native environment still struggle to reach a broad applicability and to target physiologically relevant protein concentrations. Here, we present a comparative in vitro and in-cell double electron-electron resonance (DEER) study demonstrating that nanomolar protein concentrations are at reach to measure distances up to 4.5 nm between protein sites carrying commercial gadolinium spin labels.
Asunto(s)
Electrones , Proteínas/análisis , Espectroscopía de Resonancia por Spin del Electrón , Células HEK293 , HumanosRESUMEN
Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.
Asunto(s)
Proteínas Cdc20/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Complejos Multiproteicos/metabolismo , Biocatálisis , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Mutación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas , Huso Acromático/metabolismoRESUMEN
Eukaryotic cells treated with microtubule-targeting agents activate the spindle assembly checkpoint to arrest in mitosis and prevent chromosome mis-segregation. A fraction of mitotically arrested cells overcomes the block and proliferates even under persistent checkpoint-activating conditions. Here, we asked what allows proliferation in such unfavourable conditions. We report that yeast cells are delayed in mitosis at each division, implying that their spindle assembly checkpoint remains responsive. The arrest causes their cell cycle to be elongated and results in a size increase. Growth saturates at mitosis and correlates with the repression of various factors involved in translation. Contrary to unperturbed cells, growth of cells with an active checkpoint requires Cdh1. This peculiar cell cycle correlates with global changes in protein expression whose signatures partly overlap with the environmental stress response. Hence, cells dividing with an active checkpoint develop recognisable specific traits that allow them to successfully complete cell division notwithstanding a constant mitotic checkpoint arrest. These properties distinguish them from unperturbed cells. Our observation may have implications for the identification of new therapeutic windows and targets in tumors.
Asunto(s)
Puntos de Control del Ciclo Celular , Mitosis/fisiología , Proteínas de Ciclo Celular/genética , División Celular , Proliferación Celular , Tamaño de la Célula , Perfilación de la Expresión Génica , Modelos Biológicos , Mutación , Análisis de la Célula Individual , TranscriptomaRESUMEN
Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.
Asunto(s)
Electroporación , Imagen Molecular , Proteínas Recombinantes/metabolismo , Línea Celular , Cromosomas Humanos/metabolismo , Farnesiltransferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hidrodinámica , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Mutación/genética , PrenilaciónRESUMEN
In budding yeast, the Rad9 protein is an important player in the maintenance of genomic integrity and has a well-characterised role in DNA damage checkpoint activation. Recently, roles for different post-translational histone modifications in the DNA damage response, including H2A serine 129 phosphorylation and H3 lysine 79 methylation, have also been demonstrated. Here, we show that Rad9 recruitment to foci and bulk chromatin occurs specifically after ionising radiation treatment in G2 cells. This stable recruitment correlates with late stages of double strand break (DSB) repair and, surprisingly, it is the hypophosphorylated form of Rad9 that is retained on chromatin rather than the hyperphosphorylated, checkpoint-associated, form. Stable Rad9 accumulation in foci requires the Mec1 kinase and two independently regulated histone modifications, H2A phosphorylation and Dot1-dependent H3 methylation. In addition, Rad9 is selectively recruited to a subset of Rad52 repair foci. These results, together with the observation that rad9Delta cells are defective in repair of IR breaks in G2, strongly indicate a novel post checkpoint activation role for Rad9 in promoting efficient repair of DNA DSBs by homologous recombination.
Asunto(s)
Proteínas de Ciclo Celular/química , Reparación del ADN , Histonas/química , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Cromatina/metabolismo , ADN/química , Daño del ADN , Metilación de ADN , Proteínas Fluorescentes Verdes/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lisina/química , Metilación , Proteínas Nucleares/química , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 and BubR1. A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. Here, we asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Huso Acromático/metabolismoRESUMEN
Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.
Asunto(s)
Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Línea Celular , Centrómero/química , Proteína A Centromérica/química , Proteínas Cromosómicas no Histona/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/química , Humanos , Cinetocoros/química , Cinetocoros/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αß'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.
Asunto(s)
Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Huso Acromático/metabolismo , Huso Acromático/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Dineínas/metabolismo , Células HeLa , Humanos , Cinetocoros/fisiología , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Microtúbulos/metabolismo , Mitosis/fisiología , Transporte de Proteínas/fisiologíaRESUMEN
To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Cinetocoros/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Huso Acromático/metabolismo , Línea Celular Tumoral , Células HeLa , Humanos , Mitosis/fisiología , Transducción de Señal/fisiología , Huso Acromático/fisiologíaRESUMEN
During mitotic arrest induced by microtubule targeting drugs, the weakening of the spindle assembly checkpoint (SAC) allows cells to progress through the cell cycle without chromosome segregation occurring. PLK1 kinase plays a major role in mitosis and emerging evidence indicates that PLK1 is also involved in establishing the checkpoint and maintaining SAC signalling. However, mechanistically, the role of PLK1 in the SAC is not fully understood, with several recent reports indicating that it can cooperate with either one of the major checkpoint kinases, Aurora B or MPS1. In this study, we assess the role of PLK1 in SAC maintenance. We find that in nocodazole-arrested U2OS cells, PLK1 activity is continuously required for maintaining Aurora B protein localisation and activity at kinetochores. Consistent with published data we find that upon PLK1 inhibition, phosphoThr3-H3, a marker of Haspin activity, is reduced. Intriguingly, Aurora B inhibition causes PLK1 to relocalise from kinetochores into fewer and much larger foci, possibly due to incomplete recruitment of outer kinetochore proteins. Importantly, PLK1 inhibition, together with partial inhibition of Aurora B, allows efficient SAC override to occur. This phenotype is more pronounced than the phenotype observed by combining the same PLK1 inhibitors with partial MPS1 inhibition. We also find that PLK1 inhibition does not obviously cooperate with Haspin inhibition to promote SAC override. These results indicate that PLK1 is directly involved in maintaining efficient SAC signalling, possibly by cooperating in a positive feedback loop with Aurora B, and that partially redundant mechanisms exist which reinforce the SAC.